VDU1 is a recently described ubiquitin isopeptidase that binds to and is ubiquitinated by the E3 ligase complex of the vonHippel-Lindau protein (pVHL). VDU1 has been found to be a target of ubiquitination (and proteasomal degradation) by the tumor suppressor protein complex VCB-CUL2 (an E3 ubiquitin ligase complex consisting of the von Hippel-Lindau protein pVHL, elongin C, elongin B, and cullin-2 (Li, Na et al. 2002; Li, Wang et al. 2002). pVHL is mutated in certain cancers and behaves as a tumor suppressor gene. Biochemical and genetic evidence suggests that VDU1, along with a limited number of other ubiquitination targets of the pVHL E3 ligase, has a role in establishing or maintaining the transformed state of pVHLmutant tumors. One of the E3 ligase's identified target proteins, HIFa, is known to induce angiogenic factors under certain conditions, and thus acts as an oncogene product. The beta-domain region of pVHL, which is the site of naturally occurring mutations, is the locus of VDU1 interaction, and VDU1 can be coimmunoprecipitated in the VCB-CUL2 complex. Moreover, the ubiquitination and degradation of VDU1 by a pVHL-dependent pathway is abrogated by VHL mutations that disrupt interactions with VDU1. Thus, targeted degradation of VDU1 by pVHL may be important in suppressing tumor formation and/or maintenance, and VDU1 may have oncogenic activity that is uncovered in the absence of the functional ligase. In this proposed one-year Phase I, we will configure a novel isopeptidase assay for VDU1 and configure it for high throughput screening, with the aim using this assay to discover novel antitumor drugs that act by inhibiting the cellular activity that would normally be prevented by wild type pVHL. We will also screen VDU1 against other peptide substrates to assess its cleavage patterns. The ultimate commercial goal of the development of an assay for VDU1 is to discover a drug with efficacy as a single agent or a component of conjoint therapy against renal and other cancers. An assay for the related and highly homologous isopeptidase VDU2 will be developed in Phase II and hits from the VDU1 assay screened with this assay as well.